This invention relates to a method for determining which antifungal agents are effective against a particular fungus and quantifying the concentration at which the antifungal agent is effective.
The susceptibility of fungi to antifungal agents is rarely tested routinely in hospitals, clinics or doctors' offices because of the time consuming procedures, often taking several days, and difficulties related to the stability of the antifungal agents. With present testing methods, it is necessary to make fresh solutions of nutrient or growth media to add to the agar or other non-nutrient support medium which is inoculated with the fungi. This is time consuming, expensive and tedious. Although susceptibility testing with antifungal agents is similar to that with antibacterial agents, there are certain differences which make testing with antifungal compounds more difficult.
Major difficulties include the growth characteristics of fungi in comparison to bacteria and the basic properties of the anti-fungal agents to be tested. Different antifungal agents have different solubilities, reactions to light and heat, etc. Some fungi require specialized types of growth media. In testing the susceptibility of different fungi to various types and concentrations of antifungal agents, generalized mycological growth media may be used, but must be chosen carefully to avoid inactivating the antifungal agent or agents tested.
Two of the main chemotherapeutic agents available in the United States for treatment of serious or systemic mycotic infections in man include amphotericin B and nystatin, both polyenes. The chemical properties of these compounds present problems relating to in vitro susceptibility testing. They are light-sensitive and subject to thermal destruction upon incubation. In addition, they are insoluble in water and unstable in the presence of acid. Therefore, additional precautions in using and testing these antifungal agents are necessary in comparison to many of the common antibacterial agents.
The closest prior art known to applicant is a method of testing antibiotic susceptibility of bacteria by incorporating a growth medium with agar to form nutrient agar in a petri dish. The nutrient agar is inoculated with bacteria and disks impregnated with various antibiotics of different concentration are placed on the inoculated nutrient agar. This method is relatively successful because specific growth media are usually not required for particular bacteria. Such is not the case with fungi, since the choice of growth media may be more dependent on the fungus and antifungal agent being tested.
A faster growth rate of fungi is achieved in the present invention by impregnating the disks with growth medium, rather than mixing the growth medium with the agar as in the prior art. Furthermore, the present invention avoids the necessity of mixing fresh growth medium with the agar each time a test is desired.
U.S. Pat. No. 2,986,497 of Pagano et al. discloses a method for assaying microorganisms comprising inoculating a nutrient and agar mixture contained in a petri dish with the organism to be tested. A sheet of bibulous material having a plurality of spaced apertures is impregnated with a "microorganism-affecting substance" in areas peripheral to at least one aperture. The sheet is then placed on the inoculated nutrient and agar, and the presence and amount of growth is noted. This method has the disadvantage that the nutrient medium must be made up and mixed with the agar at the time it is used. Further, it would be difficult to calculate the quantity of the microorganism-affecting substance present around each aperture of each sheet so that the procedure has limited quantitative value.
U.S. Pat. No. 3,216,907 of Goldman discloses a method for detecting microbial sensitivity to antimicrobial agents. Paper strips impregnated with a special nutrient liquid containing a carefully adjusted trace amount of glucose are required. A antimicrobial agent is applied only to the bottom of the strip or to the bottom of a petri dish. The organism to be tested is placed on top of the strip followed by the addition of one drop of water. After a very brief period of incubation, an inspection is made to determine whether the trace quantity of glucose is present, appreciably diminished or absent. Three control strips are necessary to determine whether the proper trace amount of glucose is present in each of the test strips. If not, the test must be tried again. This has a disadvantage of requiring the precise adjustment of a trace amount of glucose. Another disadvantage is that microorganisms utilize glucose or other metabolite at varying rates, so that an indirect measurement of the amount of glucose utilized is not likely to be very accurate.
U.S. Pat. No. 3,416,998 of Streitfeld discloses the use of transparent disks cut from dried agar sheets and impregnated with any of several testing agents, which may include a drug. Nutrient agar is placed in a petri dish and inoculated with the microorganism used in the test. It is necessary to prepare the nutrient medium to be mixed with the agar shortly prior to the tests. A disk made of the same nutrient agar used in the petri dish and containing the test agent is placed upon the inoculated nutrient agar. Alternatively, the organism may be rubbed on the disk. After an incubation period, the presence or absence of growth is noted. At column 7, lines 40-42, it is stated:
"It is important to note that the agar sheets of the present invention are thin dry brittle sheets incapable of supporting bacterial growth per se." PA1 "Diffusion disks are not available in this country for in vitro testing with any of the three agents, although studies are underway to permit application of the diffusion disk method to susceptibility testing with 5-FC."
In the present case, the disks are impregnated with various types of antifungal agents in various concentrations and specific nutrient medium for the particular fungus and antifungal agent being tested. The disks are placed onto non-nutrient agar inoculated with the fungus being tested. Thus, the disks of the present invention are capable of supporting fungal growth per se. In the present invention, the nutrient medium may be stored in a dry condition along with the antifungal agent on the disks.
U.S. Pat. No. 3,509,026 of Sanders discloses a process of testing the sensitivity of bacteria to antibiotics and a test element for use in the process. The test element comprises an inert disk impregnated with a particular antibiotic, nutrient medium and a substrate. The substrate is capable of detecting certain enzymes produced by the bacteria to provide a detectable change which indicates the extent of bacterial growth in the nutrient medium carried by the disk.
The present invention differs from U.S. Pat. No. 3,509,026 in that the present invention is not based on the concept of testing for the production by the organism of a particular enzyme which is detected by a substrate impregnated in the disk. The disks of the present invention contain only an antifungal agent and a nutrient medium for the fungus which is compatible with the antifungal agent being tested. The problems inherent in the process of U.S. Pat. No. 3,509,026 are avoided by the present invention. Those problems include: the non-uniform production of a vital enzyme formed by all bacteria, the different lengths of time it takes different bacteria to form a particular enzyme, and the possible interference of different enzymes or other metabolites formed by the growth of the tested bacteria.
M. Huppert et al., "Rapid Methods For Identification Of Yeasts", Journal Of Clinical Microbiology, Vol. 2, No. 1, July, 1975, pp. 21-34, disclose a method for identifying yeasts. The method includes the steps of swabbing the yeast to be identified onto hardened agar in petri dishes, allowing the streaked plates to dry, applying disks impregnated with appropriate growth media, incubating at room temperature and periodically checking the system for color changes. By testing for carbohydrate assimilation, nitrate assimilation, fermentation, urease production, pseudogerm production, production of hyphae or pseudohyphae, etc., the identity of the yeast may be determined. No antifungal agents whatsoever are used in this method.
The preparation of the nutrient media, the antifungal agents and the fungi to be tested in the present invention are similar to those reported in S. Shadomy and A. Espinel-Ingroff, "Susceptibility Testing Of Antifungal Agents", Ch. 63, pp. 569-574 in E. H. Lennett, E. H. Spaulding, and J. P. Truant, Eds., Manual Of Clinical Microbiology, 2nd Ed., American Society for Microbiology, Washington, D.C. (1974). This publication describes the susceptibility testing of antifungal agents, namely, amphotericin B, nystatin and 5-fluoro-cytosine (5-FC), by the "broth dilution method" and the "semisolid agar dilution method". At pages 570-571, it is stated:
Thus, this publication teaches away from the use of the disk method of susceptibility testing of antifungal agents according to the present invention.